Abstract Expression of the human respiratory syncytial virus (RSV) fusion protein (F) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter was analyzed by enzyme-linked immunosorbent assay (ELISA) in polyethylene glycol-transfected apple leaf pro- toplasts. In particular, we examined whether RSV-F gene expression could be enhanced by addition of a viral leader and a plant enhancer to the chimeric gene construct. Inser- tion of the 5′-untranslated leader from alfalfa mosaic virus (AMV) RNA 4 between the CaMV 35S promoter and the RSV-F gene increased viral expression by 5.5-fold com- pared to the construct without the leader. The addition of a transcriptional enhancer from the pea plastocyanin gene (PetE) upstream of the CaMV 35S promoter to a con- struct containing the AMV leader further increased RSV-F gene expression by 1.4-fold. Immunoblot assays showed that the RSV-F expressed in transfected apple pro- toplasts reacted with RSV-F monoclonal antibodies and was of the expected molecular mass of 68 kDa. These re- sults demonstrated that the RSV-F recombinant protein was expressed in an antigenic form in plant cells. Further- more, protein expression was enhanced by modifying the transfection vector using both a leader and an enhancer linked to a promoter. Key words RSV-F expression · Apple protoplasts · ELISA · Immunoblotting Abbreviations AMV Alfalfa mosaic virus · CaMV Cauli- flower mosaic virus · ELISA Enzyme-linked immunosor- bent assay · PEG Polyethylene glycol · RSV-F Respiratory syncytial virus-F protein Introduction Plants are being exploited to express recombinant proteins of pharmaceutical value. Of special note is the develop- ment of transgenic plants to deliver antigens in an edible vaccine form. Such vaccines require no secondary process- ing or purification and can be used for oral immunization. This approach is being applied to develop vaccines against pathogenic microbial antigens, for example, the capsid protein of Norwalk virus (causal agent of acute gastroen- teritis in humans) (Mason et al. 1996), the binding subunit of Escherichia coli heat-labile enterotoxin produced by en- terotoxigenic E. coli (causal agent of diarrhea) (Haq et al. 1995; Tacket et al. 1998), and the cholera toxin B subunit pentamer produced by Vibrio cholerae (causal agent of cholera) (Arakawa et al. 1998). Respiratory syncytial virus (RSV) is an RNA virus of the genus Pneumovirus containing two main antigenic pro- teins, F and G (Hall 1992). This virus is a major human pathogen and a leading cause of bronchiolitis and pneu- monia in infants, children, adults and the elderly (Hall 1992). A major difficulty in developing an effective RSV vaccine has been the fact that natural infection confers, at most, only temporary protection against the disease (Hall 1992). A viable option is to develop an edible vaccine against RSV. If the vaccine was presented in fruit, apple for example, this would represent a novel approach to de- livering the antigen orally to human mucosal tissues where it would be anticipated to produce an immune response. To express the recombinant RSV-F protein at high lev- els in plant cells, the coding sequence must be under the Plant Cell Reports (1999) 18: 394–397 © Springer-Verlag 1999 Received: 28 April 1998 / Revision received: 11 August 1998 / Accepted: 28 August 1998 J. S. Sandhu · M. D. Osadjan · S. F. Krasnyanski L. L. Domier · S. S. Korban · D. E. Buetow Enhanced expression of the human respiratory syncytial virus-F gene in apple leaf protoplasts Communicated by W. Parrott J. S. Sandhu · M. D. Osadjan 1 · D. E. Buetow () Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL 61801, USA E-mail: d-buetow@uiuc.edu Fax: +1-217-3331133 S. F. Krasnyanski · S. S. Korban Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL 61801, USA L. L. Domier United States Department of Agriculture/Agriculture Research Service and Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA Present address: 1 Department of Environmental, Population and Organismic Biology, University of Colorado, Boulder, CO 80309, USA